Recombinant adenylate cyclase toxin of bordetella induces t cell responses against tumoral antigens

ABSTRACT

An immunogenic composition comprising a recombinant protein comprising a  Bordetella  CyaA, or a fragment thereof, and a peptide that corresponds to a tumor antigen is provided as a cancer treatment. Methods of treatment with this immunogenic composition are also provided. In an embodiment, the therapeutic composition is a treatment for melanoma, and comprises epitopes from the HLA*0201 epitope. These epitopes include Tyr or GnT-V, and are present in the recombinant proteins CyaA-E5-Tyr and CyaA-E5-GnT-V.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims the benefit of priority of U.S. ProvisionalApplication No. 60/523,632 (attorney docket number 03495-6095), filedNov. 21, 2003, which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a recombinant adenylate cyclase toxin ofBordetella which induces T cell responses against tumoral antigens.

BACKGROUND OF THE INVENTION

This invention relates to compositions and methods for treating cancers.

In many animal tumor models, T cells play an important role in tumorrejection. A variety of tumor antigens recognized by CD4⁺ or CD8⁺ tumorreactive T cells have been identified on both murine and human tumors(1). CD8⁺ cytotoxic lymphocytes (CTL) are of particular interest becausethese cells specifically recognize tumor cells and kill them. Therefore,an important goal in cancer immunotherapy is to activate tumor-specificCTL.

Study of antigens recognized by CD8⁺ T cells on human melanoma hasidentified several MHC-restricted tumor epitopes that correspond tononmutated or mutated peptides derived from various self proteins (2).Several of these peptides are derived from nonmutated differentiationproteins such as tyrosinase, Melan-A/Mart-1, and gp100. These proteinsare specifically expressed in most melanocytes/melanomas, and thus, theHLA-restricted epitopes are presented by most melanoma cells frompatients expressing the relevant HLA molecules. Therefore, theseantigens could be the targets of immunotherapeutic strategies that arebased on immunization against tumor epitopes.

Other antigens expressed on tumor cells have also been described, forexample, a peptide derived from an intron sequence of the gene thatcodes for N-acetylglucosaminyl-transferase V (GnT-V) (3). This intron isspecifically expressed in melanoma cells and is present in about 50% ofmelanoma cells.

Various vaccination protocols designed to induce specific anti-tumor CTLresponses against these epitopes have been developed, includingprotocols that use free peptide in IFA (4), recombinant viral vectors(5-7), or dendritic cells (8-11). The application of these approaches tohuman vaccination remains limited due to potential toxicity ofadjuvants, bias towards the response against vector-derived epitopicpeptide, or because they are “labor-demanding” (in vitro manipulatedDC).

Previously, recombinant plasmids have been used for the expression ofBordetella sp. adenylate cyclase (cyaA) and a heterologous DNA insertedin a permissive site of CyaA. These plasmids and resulting recombinantproteins have been useful for inducing immune responses. The immuneresponses elicited have been in CD8+ T lymphocytes with class I majorhistocompatibility complexes, as well as in CD4+ T lymphocytes withclass II major histocompatibility complexes. (See U.S. Pat. Nos.5,503,829, 5,679,784, and 5,935,580.) More specifically, the recombinantproteins can be delivered to CD11b expressing cells, such as dendriticcells. (See European Patent Application EP1 188 446 A1, “Proteinaceousvectors for molecule delivery to CD11b expressing cells”, and WO/2122169A2 “Vectors for Molecule Delivery to CD11b Expressing Cells”,corresponding to U.S. Pat. No. 387,486, and European Patent ApplicationNo. 03291486.3, “Modified Bordetella Adenylate Cyclase Comprising orLacking CD11b/CD18 Interaction Domain and Uses thereof”.) See also,El-Azami-El-Idrissi, et al., 2003, Interaction of Bordetella pertussisAdenylate Cyclase with CD11b/CD18, J. Biol. Chem., vol. 278, pp.38514-21.

There exists a need in the art for new antitumor treatments that allowfor specific targeting to immune cells and T cell responses. These newstrategies should result in specific amplification of immune responsesagainst tumoral antigens.

BRIEF SUMMARY OF THE INVENTION

This invention aids in fulfilling the needs in the art by providingrecombinant CyaA proteins that induce immune responses. These responsescan be directed towards tumoral antigens.

The invention provides novel methods of treating and immunomonitoringcancers.

The invention provides an immunogenic composition comprising arecombinant protein, wherein the recombinant protein comprises aBordetella adenylate cyclase (CyaA) and a peptide that corresponds to atumor antigen.

An embodiment of the invention is a method of treating a patient withcancer comprising (1) administering an immunogenic composition to thepatient, wherein the immunogenic composition comprises a recombinantprotein, wherein the recombinant protein comprises a Bordetella CyaA ora specific fragment thereof, and a peptide that corresponds to a tumorantigen, and (2) inducing an immune response, such as a T cell response,in the patient.

An embodiment of the invention is a method of treating a patient withcancer comprising (1) administering an immunogenic composition to thepatient, wherein the immunogenic composition comprises a vectorexpressing a recombinant protein, wherein the recombinant proteincomprises Bordetella CyaA or a specific fragment thereof, and a peptidethat corresponds to a tumor antigen, and (2) inducing a T cell responsein the patient.

The T cell response is a CTL response or a T helper response or a CTLand a T helper response.

In an embodiment of the invention, the tumor is a melanoma.

In another embodiment of the invention, the tumor antigen is an HLA*0201epitope.

Encompassed in the invention is the recombinant protein is CyaA-E5-Tyror CyaA-E5-GnT-V.

In a further embodiment of the invention, the recombinant proteincomprises more than one tumor antigen. In a particular embodiment, atleast one tumor antigen is different from the other(s).

The tumor antigen is localized to any permissive site of CyaA.

In an embodiment of the invention CyaA is from Bordetella pertussis,Bordetella parapertussis, or Bordetella bronchiseptica. In a preferredembodiment CyaA is from Bordetella pertussis.

The invention also provides for an immunogenic composition comprising arecombinant protein, wherein the recombinant protein comprises at leastone specific fragment of the adenylate cyclase protein that isrecognized as a ligand on human and animal cells, and at least oneepitope specific for a tumoral antigen. In the recombinant protein ofthe immunogenic composition CyaA and the tumoral antigen can either begenetically fused or chemically bound (PCT/EPO1/11315).

Furthermore, the invention provides a recombinant protein wherein therecombinant protein comprises Bordetella CyaA, or a specific fragmentthereof, and a peptide that corresponds to an antigen comprising theGnTV epitope. The antigen is either fused or chemically bound to theCyaA protein or a specific fragment thereof.

The invention also provides a nucleic acid sequence coding for a fusionprotein comprising Bordetella CyaA, or a specific fragment thereof, anda peptide that corresponds to an antigen comprising the GnTV epitope. Ina particular embodiment, said sequence is included in the plasmiddeposited at C.N.C.M., Paris, France, on Oct. 16, 2003 under accessionnumber I-3111.

Also included in the invention is a vector expressing a recombinantprotein which comprises Bordetella CyaA, or a specific fragment thereof,and a peptide that corresponds to an antigen comprising the GnTVepitope. In a particular embodiment, said vector has been deposited atC.N.C.M., Paris, France, on Oct. 16, 2003 under accession number I-3111.

The invention further encompasses a host cell that expresses arecombinant protein comprising Bordetella CyaA, or a specific fragmentthereof, and a peptide that corresponds to an antigen comprising theGnTV epitope. In a particular embodiment, the host cell expresses thevector that has been deposited at C.N.C.M., Paris, France, on Oct. 16,2003 under accession number I-3111.

The invention also provides a nucleic acid sequence coding for a fusionprotein comprising Bordetella CyaA, or a specific fragment thereof, anda peptide that corresponds to an antigen comprising the Tyr epitope. Ina particular embodiment, said sequence is included in the plasmiddeposited at C.N.C.M., Paris, France, on May 31, 2001 under accessionnumber I-2679.

Another embodiment of the invention is a vector expressing a recombinantprotein that comprises Bordetella CyaA, or a specific fragment thereof,and a peptide that corresponds to an antigen comprising the Tyr epitope.In a particular embodiment, said vector has been deposited at C.N.C.M.,Paris, France, on May 31, 2001, under accession number I-2679.

The invention further encompasses a host cell that expresses arecombinant protein comprising Bordetella CyaA, or a specific fragmentthereof, and a peptide that corresponds to an antigen comprising thepTyr epitope. In a particular embodiment, the host cell expresses thevector that has been deposited at C.N.C.M., Paris, France, on May 31,2001, under accession number I-2679.

DESCRIPTION OF THE DRAWINGS

This invention will be described with reference to the drawings inwhich:

FIG. 1 depicts in vivo induction of CTL responses by recombinant CyaAcarrying HLA*0201 restricted melanoma epitopes. HHD-mice received i.p.injections on days 0, 21 and 42 of either 50 μg control CyaA toxin (,◯) or recombinant CyaA toxins carrying melanoma epitopes (▪, □)(A,CyaA-Tyr; B, CyaA-GnT-V) in the presence of 1 mg alum. Seven days afterthe last injection, spleen cells from immune mice were stimulated invitro with the priming peptide pTyr (A), or pGnT-V (B) in the presenceof irradiated syngenic spleen cells. The cytotoxic activity of theseeffector cells was measured on ⁵¹Cr-labeled RMA-S-HHD target cellspulsed with the respective peptide (, ▪) or incubated with medium alone(◯, □). The data represent mean values of duplicates (SD<10%). Quadrantsrepresent the number of positive mice versus the number of tested mice,and curves represent mean values±SD of responder mice per group fromthree experiments.

FIG. 2 depicts induction of melanoma-specific CTL responses byrecombinant CyaA carrying melanoma epitopes using different routes ofimmunization. Panels A and B: HHD mice were immunized i.p. twice on days0 and 21 with 50 μg wild-type CyaA (, ◯) or recombinant CyaA-Tyr (▪, □)in the presence (A) or in the absence of 1 mg alum (B). Panels C and E:HHD mice were immunized by one i.v. injection with 50 μg control wildtype CyaA (, ◯) or recombinant CyaA-Tyr (▪, □) (C) or recombinantCyaA-GnT-V (▪, □) (E) in the absence of adjuvant. Panels D and F: HHDmice were immunized by one i.v. injection with 50 μg control detoxifiedCyaA-E5 (, ◯) or detoxified recombinant CyaA-E5-Tyr (▪, □) (D) orrecombinant CyaA-E5-GnTV (▪, □) (F) in the absence of adjuvant. Sevendays after the last injection, spleen cells from immune mice werestimulated in vitro with priming peptides in the presence of irradiatedsyngeneic spleen cells. The cytotoxic activity was measured on⁵¹Cr-labeled RMA-S-HHD target cells pulsed with the priming peptide (,▪) or incubated with medium alone (◯, □). The results show cumulativedata from 2-4 experiments. Quadrants represent the number of positivemice versus the number of tested mice, and curves represent meanvalues±SD of responder mice per group. The results obtained afterimmunization with toxic and detoxified CyaA are not statisticallydifferent using a t test.

FIG. 3 demonstrates that immunization of mice with CyaA-Tyr induces along-lasting specific memory CTL activity. HHD mice were immunized i.p.twice on days 0 and 21 with 50 μg wild-type CyaA (, ◯) or recombinantCyaA-Tyr (▪, □) in the presence of 1 mg alum. Three months (A) or fivemonths (B) after the last injection, spleens were removed and specificCTL activity was measured after in vitro stimulation as described inFIG. 1 on ⁵¹Cr-labeled RMA-S-HHD target cells pulsed with the peptidepTyr (, ▪) or incubated with medium alone (◯, □). Quadrants representthe number of positive mice versus the number of tested mice. Curvesrepresent mean values±SD of responder mice per group from oneexperiment.

FIG. 4 depicts stimulation of human specific CTL clones by humandendritic (DC) cells incubated with recombinant CyaA-E5-Tyr orCyaA-E5-GnT-V. Due to the cytotoxicity of CyaA, only detoxifiedrecombinant CyaA were tested in vitro. Immature HLA*0201⁺ DC derivedfrom human monocytes were incubated with CyaA-E5 (◯), recombinantCyaA-E5-Tyr () (A), CyaA-E5-GnT-V () (B) or with the relevantantigenic peptide (▴), and were used as APC (Antigen Presenting Cells)to stimulate anti-tyrosinase CTL clone IVS-B (A) or anti-GnT-V CTL cloneCMU 579 6/3 (B). The secretion of IFN-γ by the CTL clones was assessedby ELISA. The results are expressed as the mean concentration of IFN-γreleased in the supernatants from duplicate wells and are representativeof three independent experiments. Standard errors of the mean areindicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A new approach for CTL activation has recently been developed based onbacterial toxins capable of delivering antigenic epitopes across theplasma cell membrane into the cytosol, where appropriate processing andinteraction with MHC-class I molecules can occur. The adenylate cyclasetoxin (CyaA) of Bordetella pertussis (Glasser, P., et al. 1988Bordetella pertussis adenylate cyclase: the gene and the protein, TokaiJ. Exp. Clin. Med., 13 Supp.: 239) has the capacity to deliver itscatalytic domain into the cytosol of eukaryotic cells (12). Delivery ofa CM⁺ T cell epitope inserted into the catalytic domain of CyaA resultsin intracellular processing and presentation of the epitope by MHC-classI molecules at the surface of antigen presenting cells (13).Furthermore, CyaA specifically binds to α_(M)β₂ integrin (CD11b/CD18)(14), and thus, targets the CD11b+ DC subpopulation, which veryefficiently induces primary immune responses (15). Therefore,immunization of mice with a recombinant CyaA toxin bearing a viralepitope leads to the induction of strong CTL responses and to a fullprotection against a lethal viral challenge (16).

Moreover, CyaA toxins carrying a single CTL epitope can also stimulateefficient protective and therapeutic antitumor immunity in mice (17).Importantly, genetically detoxified CyaA toxoids retain the property toinduce protective antiviral or antitumoral immunity (17, 18). Thus, CyaAseems to be a safe and efficient non-replicating vector to inducespecific immune responses in mice. However, in the view of elaboratingcancer immunotherapy using CyaA, it is of particular importance todemonstrate that human tumoral epitopes inserted into CyaA areefficiently processed and presented in association with human MHCmolecules.

In one embodiment of the invention, two recombinant CyaA carryingHLA*0201 restricted melanoma epitopes derived either from tyrosinase orfrom GnT-V were constructed. The potency of these recombinant CyaA toinduce in vivo HLA*0201 restricted CTL responses against the insertedepitopes and the ability to deliver these epitopes to human antigenpresenting cells is demonstrated in the Examples below.

It was discovered that CyaA of Bordetella pertussis is able to deliverCD8⁺ T cell epitopes into the cytosol of CD11b⁺ dendritic cellsfollowing its specific interaction with the α_(M)β₂ integrin(CD11b/CD18). This delivery results in intracellular processing andpresentation by MHC-class I molecules of the CD8⁺ T cell epitopesinserted into CyaA. Indeed, CyaA toxins carrying a single CTL epitopecan induce efficient protective, and therapeutic antitumor immunity inmice.

It was further discovered that these recombinant CyaA proteins inducestrong anti-melanoma CTL responses in HLA*0201-transgenic mice, evenafter a single intravenous immunization without adjuvant. The responsesare long lasting, being detected as long as five months after the lastinjection.

Finally, it was discovered that human dendritic cells, treated with therecombinant CyaA, process and efficiently present melanoma epitopes tohuman CTL clones. The recombinant CyaA proteins of the inventiondemonstrate that tumoral epitopes inserted into CyaA are efficientlyprocessed and presented in association with human MHC molecules.Therefore, CyaA is capable of activating antitumoral CTL in humans, andis a novel factor for cancer immunotherapy.

As used herein, the term “immunogenic composition” relates to acomposition that leads to an immunological response and that isassociated with therapeutic treatments, such as treatments againstcancers.

As used herein the terms “Bordetella CyaA” or “Bordetella adenylatecyclase” encompass the CyaA or a fragment thereof, either modified ornot. The modifications can include deletion of some internal aminoacids. For example, CyaA may have no catalytic activity, but thespecific binding to CD11b/CD18 receptor and the process of translocationof the catalytic domain are not affected. The term “Bordetella” refersto the adenylate cyclase protein of a pathogen of Bordetella species.Said pathogen can be Bordetella pertussis, Bordetella parapertussis, orBordetella bronchiseptica.

As used herein, the term “antigen” or “epitope” refers to a peptideincluding a protein that can induce an immune response. The term“heterologous” refers to the nature of the antigen bound to the CyaAprotein, which induces an immune response different from that of theCyaA protein. A heterologous antigen or epitope can be fused to CyaA orchemically bound to CyaA, for instance.

As used herein, the term “immunogenic” refers to a characteristic of aprotein as being able to elicit an immune response in a mammal,particularly in a human. The term “immune response” refers to manyeffects that are caused by cells of the immune system, such as, forinstance, a CTL response and/or a T helper response, and in the contextof the invention includes, but are not limited to, activation oftumor-specific cytotoxic lymphocytes. As used herein, the term“immunotherapy” refers to a therapy for a disease that relies on animmune response.

In addition to the recombinant protein or vector of the invention, theimmunogenic composition of the invention can include adjuvants andexcipients to allow an increase and modulation in the immune response.These adjuvants are diverse in nature. They can, for example, compriseliposomes, oily phases, for example, the Freund type of adjuvants, whichare generally used in the form of an emulsion with an aqueous phase, or,more commonly, can comprise water-insoluble inorganic salts. Theseinorganic salts can comprise, for example, aluminum hydroxide, zincsulfate, colloidal iron hydroxide, calcium phosphate, or calciumchloride. Aluminum hydroxide (Al(OH)₃) is the most commonly usedadjuvant.

The invention also encompasses recombinant proteins comprisingBordetella CyaA, or a specific fragment thereof, and the peptide pTyr(YMDGTMSQV). Said peptide may comprise extended flanking sequences. ThepTyr peptide corresponds to the melanoma HLA*0201 restricted epitopefrom the 369-377 region of tyrosinase. Note that the amino acids 369-377of human tyrosinase are YMNGTMSQV. However, the Asn residue at position371 (N) of tyrosinase is naturally deamidated to Asp (D) of this epitopein living cell so that the true epitope recognized by the CTL clones invivo are recognizing sequence YMDGTMSQV.

Furthermore, an epitope with an extended flanking sequence of amino acid360-385 of human Tyrosinase is SSMHNALHIYMNGTMSQVQGSANDPI (SEQ ID NO: 1)(with N371 converted to D).

An epitope derived from the human N-acetylglucosaminyltransferase V geneis VLPDVFIRC (Y Guilloux, et al. A peptide recognized by human cytolyticT lymphocytes on HLA-A2 melanomas is encoded by an intron sequence ofthe N-acctylglucosaminyltransferase V gene, J. Exp. Med. 1996 183:1173-1183.) The Gnt-V epitope is encoded by an intron sequence that maycode for a 74 amino acid long polypeptide (H. sapiens DNA for exonencoding for N-acetylglucosaminyltransferase V; Accession #X91652).Furthermore, the epitope with extended flanking sequences from humanN-acetylglucosaminytransferase V is MVLPDVFIRCVVFCL (SEQ ID NO: 2).

The invention also encompasses the recombinant fusion protein comprisingBordetella CyaA, or a specific fragment thereof, and the peptide pGnT-V(VLPDVFIRC). Said peptide may comprise extended flanking sequences. Thepeptide pGnT-V corresponds to the HLA*0201 restricted epitope NA17-Aderived from an intron of the N-acetylglucosaminyl-transferase V gene.

In one preferred embodiment of the invention the recombinant protein isCyaA-Tyr. The term “CyaA-Tyr” means a fusion protein comprising thetyrosinase melanoma epitope of HLA*0201, which can be prepared asdescribed in Example 1, and Bordetella pertussis CyaA. The term“CyaA-E5-Tyr” refers to the CyaA-Tyr protein in which the catalyticactivity of CyaA has been genetically inactivated. See, for instance,Example 1.

In another preferred embodiment of the invention, the recombinantprotein is CyaA-E5-GnT-V. The term “CyaA-E5-GnT-V” means a fusionprotein comprising the NA17-A melanoma epitope of HLA*0201 derived froman intron of the N-acetylglucosaminyl-transferase V gene, which can beprepared as described in Example 1, and Bordetella pertussis CyaA. Theterm “CyaA-E5-GnT-V” refers to the Cya-GnT-V protein in which thecatalytic activity of CyaA has been genetically inactivated. Once again,see, for instance, Example 1.

In yet another embodiment of the invention, the recombinant proteinbetween CyaA and pTyr or pGnT-V is modified from the structure ofCyaA-Tyr, CyaA-GnT-V, CyaA-E5-Tyr, or CyaA-E5-GnT-V. Modification ofthese embodiments can include the addition of flanking regions, whichare sequences of amino acids that surround the peptides comprising therecombinant protein, and were described above. These flanking sequencescan enhance processing. Flanking sequences can also be sequences whichis not naturally surround the antigen but which specifically enhance theantigen processing by antigen preventing cells.

In yet another embodiment, the recombinant proteins can be modified byincluding multiple identical heterologous epitopes. For instance, Tyror. GnT-V epitope, as described above, or other melanoma epitopes.

In further embodiments of the invention, the recombinant protein caninclude at least one specific fragment of the adenylate cyclase protein,such as, but not limited to, CyaA the 373-1706 region or the 1166-1281region which are recognized as a ligand on human and animal cells, suchas, dendritic cells, and at least one epitope specific for a cancerantigen, such as, but not limited to, pTyr or GnT-V.

In another embodiment of the invention, the recombinant protein caninclude multiple epitopes from one or more tumoral antigens.

Another embodiment of the invention includes permissive sites of CyaAthat differ from those provided in the Examples. The antigen portion ofthe recombinant protein used in the tests of the invention can belocalized to any permissive site of the CyaA adenylate cyclase proteinWO 93/21324. In addition, the invention encompasses tests andimmunogenic compositions that utilize only fragments of the CyaAadenylate cyclase in the recombinant protein (see EPO 03/291486.3).

As used herein, the term “permissive site” relates to a site where theheterologous peptide can be inserted without substantially affecting thedesired functional properties of the adenylate cyclase toxin, i.e.without affecting the domains necessary for the specific binding toCD11b/CD18 receptor and advantageously without affecting the process oftranslocation of the catalytic domain.

Permissive sites of the Bordetella pertussis adenylate cyclase include,but are not limited to, residues 137-138 (Val-Ala), residues 224-225(Arg-Ala), residues 228-229 (Glu-Ala), residues 235-236 (Arg-Glu), andresidues 317-318 (Ser-Ala) (see Sebo et al., 1985). The followingadditional permissive sites are also included in embodiments of theinvention: residues 107-108 (Gly-His), residues 132-133 (Met-Ala),residues 232-233 (Gly-Leu), and 335-336 (Gly-Gln) and 336-337. (Seegenerally, Glaser et al., 1988 Bordetella pertussis adenylate cyclase:the gene and the protein, Tokai J. Exp. Clin. Med., 13 Suppl.: 239-52.)

As used herein, the terms “specific region of the adenylate cyclaseprotein” or “fragment of the CyaA adenylate cyclase” relates to afragment of said protein including the protein wherein some amino acidswhich are not on the tumoral parts of the protein have been deleted, andthe desired functional properties of adenylate cyclase toxin are notsubstantially affected, i.e. the domains necessary for the specificbinding to CD11b/CD18 receptor and the process of translocation of thecatalytic domain are not affected.

The terms “tumor antigen” or “cancer antigen” refer to any substancefrom a tumor that elicits an immune response and reacts specificallywith antibodies or T cells. Said substance can be from any origin,either spontaneous or from a virus, which transforms cells to form atumor. Examples of such viruses are HHV8, HCV, and HBV. The antigen orepitope must be present on the surface of the tumor cell.

As used herein, the term “a peptide that corresponds to an antigen”encompass an antigen, an epitope, or an antigen or an epitope flanked bynaturally or non-naturally occurring flanking regions, whichspecifically enhance antigen processing by antigen presenting cells.

The term “epitope” refers to the minimal peptide sequence of an antigenthat can induce an immune response.

The term “peptide” refers to a series of amino acids linked by amidebonds, comprising at least 3 amino acids, and preferably at least 6amino acids.

The immunogenic composition of the invention can be used in solution,for example, but not limited to, in PBS, or with adjuvants, for example,but not limited to alum. The immunogenic composition can be administeredintramuscularly, subcutaneously, intravenously, or intradermally. Theimmunogenic composition can be administered in amounts from 0.5-10 mg,preferably 1-5 mg, 1.5-3 mg, or more preferably 1.50 mg. The effects ofthese treatments can be monitored by assaying the levels of IFN-γ withELISPOT, ELISA, or CTL activation assays, or other appropriateimmunoassays.

Publications illustrate the use of recombinant adenylate cyclase ofBordetella sp. for diagnosis and immunomonitoring, i.e., Vordermeier H.Martin et al (Infection and Immunity, November 2004, p. 6255-2261) andSchlecht G. et al (The Journal of Immunology 2004, p. 6089-6097). Aseparate US patent application is filed by Leclerc et al. on the sameday as the present application is filed concerning “RecombinantAdenylate Cyclase of Bordetella sp. For Diagnostic and ImmunomonitoringUses, Method of Diagnostic or Immunomonitoring Using Said RecombinantAdenylate Cyclase, Kit for Diagnosing or Immunomonitoring Comprisingsaid Recombinant Adenylate Cyclase” (Attorney Docket No. 03495-0326) andclaiming priority to U.S. Provisional Application 60/523,704, filed Nov.21, 2003. These references and application are hereby incorporated byreference as regards the use of recombinant adenylate cyclase ofBordetella sp. for diagnosis and immunomonitoring.

The adenylate cyclase of Bordetella Sp. represents a new delivery systemable to specifically stimulate CD8⁺ T lymphocytes leading to protectiveantiviral and antitumoral immunity in mice (16, 17). CyaA is a powerfulnon-replicating vector for induction of adaptive immunity and is usefulin vaccines. Demonstration, according to this invention, that theinserted epitopes can be processed and presented in association withhuman MHC molecules is an indispensable prerequisite for the use of thisvector in humans.

By using recombinant CyaA in which human melanoma epitopes expressingthe human HLA*0201 class I molecule were present, strong and lastingmelanoma specific CTL responses could be induced in HLA transgenic mice.Similar results were obtained with recombinant detoxified CyaA devoid ofadenylate cyclase activity. CyaA represents an efficient vector toinduce specific CTL responses in vivo because more than 80% of immunizedHHD mice responded to the tyrosinase epitope inserted into CyaAfollowing one i.v. injection without adjuvant, while only 26% of HHDmice respond to this epitope following one injection of 100 μg ofpeptide in the presence of IFA (partially in 26). In addition, it wassurprisingly observed according to this invention that human DCefficiently processed these recombinant molecules for antigenic peptidepresentation to human CTL. Strikingly, the recombinant CyaA-Tyr was muchmore efficient than the synthetic peptide in delivering the tyrosinaseepitope to DC.

Alternative antigen delivery systems based on recombinant virusesusually result in an in vitro presentation efficiency that is lower thanthe synthetic peptide. The surprising results from in vivo and in vitroexperiments according to the invention underline the power of CyaA asdelivery system, and show that CTL responses can be obtained in humansafter immunization with recombinant CyaA and thus, that efficientimmunotherapy can be achieved with this vector. However, theimmunogenicities of the two recombinant CyaA tested in this study werequite different. Indeed, strong CTL responses in HHD mice were inducedwith only one i.p. injection of CyaA-Tyr in the absence of adjuvant,while three i.p. injections of CyaA-GnT-V, in the presence of alum, wererequired to generate specific CTL responses. The weak efficiency ofCyaA-GnT-V to deliver GnT-V melanoma epitope was also evidenced invitro, since human DC incubated with this vector poorly stimulated ananti-GnT-V CTL clone as compared to CyaA-Tyr, which efficientlystimulated a specific anti-tyrosinase CTL clone.

This difference can be explained by the fact that the GnT-V peptidegrafted into CyaA-GnT-V was poorly processed, as compared to thetyrosinase peptide inserted into CyaA-Tyr. Indeed, flanking regions of agiven epitope are known to influence the proteolytic generation of themature peptide (27-29) and particularly for subdominant and/or crypticepitopes (30). Therefore, it is expected that modification of themolecular context of GnT-V epitope into CyaA can enhance the efficiencyof processing of this epitope by APC. On the contrary, the sequenceflanking the tyrosinase epitope in CyaA-Tyr appears to allow itsefficient processing.

Furthermore, CyaA-Tyr is very efficient in activatingHLA*0201-restricted CD8⁺ T cell in vivo, because a single intravenousimmunization or two i.p. injections without adjuvant were sufficient togenerate strong specific CTL responses. This is explained by the factthat CyaA targets specifically CD11b⁺ DC, the most potent APC to induceprimary response, as a result of its interaction with the α_(M)β₂integrin expressed by these cells (14). Thus, CyaA has the exceptionalproperty of specifically delivering antigens to the cytosolic Ag class Ipresentation pathway of professional APC.

Further improvements of the CyaA recombinant strategy are also possible.First, multiple insertions of CD8⁺ T cell epitopes into the samerecombinant molecule has already been successfully achieved. Indeed,immunization of mice with recombinant CyaA carrying three differentepitopes, including a LCMV epitope, leads to the induction of specificCTL responses for each of the three epitopes, as well as protectionagainst a lethal LCMV challenge (31). Detoxified CyaA carrying multiplemelanoma epitopes constitute a good alternative to induce multispecificCTL responses. Furthermore, additional insertion of CD4⁺ T cell epitopicpeptides is also possible. Although the implication of CD8⁺ T cells ineradication of established tumors has been clearly demonstrated (32), Thelper cells can also be required to induce efficient anti-tumoralresponses (33-35). Recombinant CyaA can also deliver epitopes into theMHC class II processing pathway (36) and is able to induce, in vivo,both specific Th1 and CTL responses (37). This characteristic is ofgreat interest for vaccination strategies where both kinds of T cellresponses have to be induced, noticeably in the context of cancerimmunotherapy.

Plasmid pTRACE5-GnTV is a derivative of the expression vector pTRACGthat expresses the cyaC and cyaA genes from Bordetella pertussis underthe control of the λ phage Pr promoter (pTRCAG also harbors anampicillin resistance selectable marker and the thermosensitive λrepressor CI⁸⁵⁷). In pTRACE5-GnTV, the cyaA gene is modified byinsertion of a dipeptide Leu-Gln between codons 188 and 189 of wild-typeCyaA (resulting in the inactivation of the adenylate cyclase activity)and by insertion of a DNA sequence encoding the following peptidesequence PASVLPDVFIRCGT (SEQ ID NO: 3) inserted between codons 224 and240 of CyaA. The underlined peptide (VLPDVFIRC) (SEQ ID NO: 4)corresponds to the HLA-A2 restricted melanoma epitope NA17-A derivedfrom the N-acetylgiucosaminyl-transferase V gene. (G. Dadaglio, et al.(2003) Recombinant adenylate cyclase of Bordetella pertussis induces CTLresponses against HLA-A2-restricted melanoma epitope. Int. Immuno.)Plasmid XL1/pTRACE5-GnTV was deposited at C.N.C.M. on Oct. 16, 2003,with accession number I-3111.

Plasmid pTRACE-5-Tyros369 is a derivative of the expression vectorpTRACG that expresses the cyaC and cyaA genes from Bordetella pertussisunder the control of the λ phage Pr promoter (pTRCAG also harbors anampicillin resistance selectable marker and the thermosensitive λrepressor CI⁸⁵⁷). In pTRACE5-Tyros369, the cyaA gene is modified byinsertion of a dipeptide Leu-Gln between codons 188 and 189 of wild-typeCyaA (resulting in the inactivation of the adenylate cyclase activity)and by insertion of a DNA sequence encoding the following peptidesequence PASYMDGTMSQVGTRARLK (SEQ ID NO: 5) inserted between codons 224and 240 of CyaA. The underlined peptide (YMDGTMSQV) (SEQ ID NO: 6)corresponds to the amino acids sequence 369-377 of tyrosinase. PlasmidXL1/pTRACE5-Tyros369 was deposited at C.N.C.M. on May 31, 2001, withaccession number I-2679.

The abbreviations used are as follows: CTL: cytotoxic T lymphocytes; DC:dendritic cells; PBMC: peripheral blood mononuclear cells; CyaA:adenylate cyclase of Bordetella sp; Tyr: tyrosinase; GnT-V:N-acetylglucosaminyl-transferase V; GM-CSF: granulocyte-macrophagecolony-stimulating factor; IFN: interferon; i.p.: intraperitoneal; i.v.:intravenous.

This invention will be described in greater detail in the followingExamples.

Example 1 Materials and Methods

Mice. HHD mice are H-2D^(−/−) β2^(−/−) double knock out mice expressingthe HHD transgene comprising the α1 (H) and α2 (H) domains of HLA*0201linked to α3 transmembrane and cytoplasmic domains of H-2D^(b) (D), withthe α 1 domain linked to human β2-microglobulin. Thus, the only MHCclass I molecule expressed by the HHD mice is the modified HLA*0201molecule (19). HHD mice were bred and housed in animal facilities ofInstitut Pasteur.

Peptides. The synthetic peptides pTyr (YMDGTMSQV) (SEQ ID NO: 6)corresponding to the melanoma HLA*0201 restricted epitope from the369-377 region of tyrosinase (20, 21) and pGnT-V (VLPDVFIRC) (SEQ ID NO:4) corresponding to the HLA*0201 restricted epitope NA17-A derived froman intron of the N-acetylglucosaminyl-transferase V gene (3) werepurchased from Neosystem (Strasbourg, France).

Construction of recombinant Bordetella pertussis adenylate cyclasetoxins and toxoids carrying melanoma epitopes. The recombinant CyaAtoxin, CyaA-Tyr, harbors a 14 amino acid long polypeptide sequence(PASYMDGTMSQVGT (SEQ ID NO: 7), one-letter code for amino acid)genetically inserted between residues 224 and 225 of CyaA. This sequencecontains a single copy of the HLA*0201 restricted melanoma epitopederived from tyrosinase (amino acid 369-377, underlined sequence above).Recombinant CyaA toxin CyaA-GnT-V harbors a 14 amino acid long(PASVLPDVFIRCGT) (SEQ ID NO: 3) insert at the same position and containsa single copy of the HLA*0201 restricted melanoma epitope NA17-A derivedfrom the N-acetylglucosaminy-transferase V gene (underlined sequenceabove).

These recombinant toxins were produced in the E. coli strain BLR(Novagen) by using expression vectors that are derivatives of the pTRACGplasmid (22), modified by the insertion between the NheI and KpnIrestriction sites of appropriate synthetic double strandedoligonucleotides encoding the indicated polypeptide sequences. The E.coli strain XL1-Blue (Stratagene) was used for all DNA manipulationsthat were done according to standard protocols. The recombinant proteinswere purified to homogeneity from inclusion bodies by a two-stepprocedure that includes DEAE-Sepharose and Phenyl-Sepharosechromatographies, as described previously (23).

The recombinant toxins CyaA-Tyr and CyaA-GnT-V are enzymatically activeand therefore cytotoxic. The recombinant toxoids CyaA-E5-Tyr andCyaA-E5-GnT-V are enzymatically inactive, detoxified variants ofCyaA-Tyr and CyaA-GnT-V respectively. They are unable to synthesize cAMPas a result of a dipeptide insertion within a critical region of thecatalytic site (23). CyaA-E5-Tyr and CyaA-E5-GnT-V toxoids were producedin E. coli by using expression vectors that are derivatives of thepTRACE5 plasmid: this plasmid was obtained by insertion of thehexanucleotide CTGCAG (SEQ ID NO: 8) in an EcoRV site located in the 5′part of the cyaA DNA sequence. This results in an in-frame insertion ofthe dipeptide Leu-Gln between Asp188 and Ile189 of CyaA (23). The samesynthetic double stranded oligonucleotides described above were insertedbetween the NheI and KpnI sites of pTRACE5 to create plasmidspTRAC-E5-Tyr and pTRAC-E5-GnT-V. The recombinant CyaA-E5-Tyr andCyaA-E5-GnT-V toxoids were purified to homogeneity as described (23).

All purified recombinant toxins and toxoids were more than 90% pure asjudged by SDS-gel analysis. The toxin concentrations were determinedspectrophotometrically from the absorption at 280 nm using a molecularextinction coefficient of 142,000 M⁻¹ cm⁻¹.

Oligonucleotide synthesis and DNA sequencing were performed by Genaxis(France). Cultures in fermentors were performed by the Service desFermentations facility from Institut Pasteur.

Mouse immunization. Six to ten week old female HHD mice were immunizedwith two or three i.p. injections at 21 day intervals of either 50 μgCyaA or recombinant CyaA carrying melanoma epitopes in the presence orin the absence of 1 mg alum. In some experiments, mice were immunizedwith one i.v. injection of 50 μg of the recombinant CyaA in PBS.Detoxified recombinant CyaA-E5 were used in the same conditions. Spleenswere surgically removed seven days after the last injection, except forthe analysis of long lasting responses, where spleens were removed threeor five months after the last injection.

In vitro cytotoxic assay. Spleen cells from immunized mice werestimulated in vitro with 10 μg/ml of pTyr or pGnT-V peptidescorresponding to the priming epitope in the presence of syngeneicirradiated naive spleen cells in complete medium (RPMI 1640 mediumcontaining L-Alanyl-L-Glutamine dipeptide supplemented with 10% fetalcalf serum, 5×10⁻⁵ M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/mlstreptomycin and 20 mM Hepes) for five days. The cytotoxic activity ofthese effector cells was tested in a 4-h ⁵¹Cr-release assay on HHDtransfected TAP^(−/−) RMA-S cells (RMA-S-HHD) loaded with the relevantpeptides as target cells. ⁵¹Cr-labeling was performed as follows: oneday before the cytotoxic test, RMA-S-HHD cells were incubated overnightat room temperature in 7% CO₂ equilibrated RPMI 1640 medium supplementedwith 20 mM Hepes. Then, cells were incubated 3 h at room temperaturewith or without 20 μg/ml of the relevant peptide, washed once andradiolabeled with 100 μCi of ⁵¹Cr for 1 h at 37° C.

Various effectors to target ratios were used and all assays were done induplicate. The radioactivity released in the supernatant of each wellwas measured. The percentage of specific lysis was calculated as100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release). Maximum release was generated by adding10% triton X-100 to target cells and spontaneous release was obtainedwith target cells incubated in medium alone. Mice are considered to beresponders when at least 20% specific lysis was observed at the highestE/T ratio. The results are expressed as mean values±SD of responder miceper group. No specific CTL activity was obtained with splenocytes fromimmunized mice stimulated in vitro with an irrelevant peptides.

Human dendritic cells. Human dendritic cells were derived from adherentmonocytes as follows: Peripheral blood mononuclear cells were isolatedfrom buffy coats obtained from the HLA*0201 hemochromatosis patientLB2050 by centrifugation on Leucosep tubes (Greiner, Frickenhausen,Germany) previously centrifuged 30 sec at 2200 rpm with 15 ml ofLymphoprep (Nycomed Pharma, Oslo, Norway). These tubes were centrifugedat 2200 rpm for 20 min at room temperature and the top part containingplasma was discarded. The interphase containing the PBMC was harvestedand washed at least three times in cold phosphate buffer with 1 mM EDTAin order to eliminate the remaining platelets. The PBMC were then leftto adhere for 1 h in culture flasks at a density of 2×10⁶ cells/cm² inRPMI 1640 supplemented with amino-acids (L-arginine 116 μg/ml,L-asparagine 36 μg/ml, L-glutamine 216 μg/ml), antibiotics, and 10%fetal calf serum (hereafter referred as culture medium). Non-adherentcells were discarded, adherent cells were washed twice carefully with 20ml of medium and incubated in culture medium with 200 U/ml human IL-4and 70 ng/ml human GM-CSF. On day two and four, 5 ml of medium wereremoved and 10 ml containing 700 ng of GM-CSF were added. IL-4 was alsosupplemented at 200 U/ml for the total volume of the flask. The cellswere used between day five and seven.

Human CTL clones. CTL clone IVS-B directed against the HLA*0201restricted Tyr epitope (positions 369-377) of tyrosinase was previouslydescribed (24). The clone was stimulated weekly with 50 U/ml human IL-2,irradiated HLA*0201 transfected MZ2-MEL melanoma cells pulsed with 2μg/ml pTyr peptide, and irradiated LG2-EBV cells as feeder cells. TheCTL clone CMU 579 6/3 specific for the HLA*0201 restricted epitopederived from GnT-V was obtained from the blood of a healthy donorfollowing a recently described method (25). Briefly, the PBMC werestimulated for two weeks with the antigenic peptide pGnT-V, human IL-2,IL-4, and IL-7. On day 13, PBMC were stained with an HLA*0201 tetramerfolded with the pGnT-V peptide. Tetramer positive lymphocytes werecloned using flow cytometry. They were stimulated for two weeks withirradiated allogeneic HLA*0201-positive EBV-transformed B cells pulsedwith the peptide, irradiated allogeneic PBL, IL-2, IL-4, and IL-7, andthen maintained by weekly stimulation with irradiated HLA*0201-positivepeptide-pulsed allogeneic tumor cells and irradiated allogeneic EBV-Bcells. Both CTL clones were maintained in Iscove's medium supplementedwith 10% of human serum, amino acids, and antibiotics.

In vitro stimulation assay of human CTL clones. For the stimulationassay, 10,000 immature dendritic cells were seeded in U-bottommicroplates in 25 μl of X-Vivo 10 medium (Whittaker Bioprodutcs,Walkersville, USA). 25 μl of CyaA preparations diluted in X-Vivo 10medium at different concentrations were added to the wells. After 30 minof incubation, the corresponding CTL clones were incubated with thesecells (75 μl of X-vivo medium containing 10⁴ anti-tyrosinase CTL cloneIVS-B or 10⁴ anti-GnT-V CTL clone CMU 579 6/3) and IL-2 (at a finalconcentration of 25 U/ml). The supernatants were collected after 20 hand their IFN-γ content was determined by ELISA (BiosourceInternational, Camarillo, Calif.). To control the ability of DCincubated with the various detoxified recombinant toxoids to stimulatethe CTL clones, they were exogenously loaded with the relevant antigenicpeptides, incubated with the relevant CTL clones and the production ofIFN-γ was similarly assessed (data not shown).

Example 2 Induction of Melanoma-Specific CTL Responses by Immunizationof HHD Transgenic Mice with Recombinant CyaA CarryingHLA*0201-Restricted Melanoma Epitopes

To determine whether the CyaA toxin is capable of inducing specific CTLresponses against human tumoral antigens, two recombinant CyaA carryingHLA*0201-restricted human melanoma epitopes were constructed. The firstrecombinant CyaA expresses the epitope 369-377 from the tyrosinaseantigen (CyaA-Tyr) and the second one expresses the epitope NA17-Aderived from an intron of the N-acetylglucosaminyl-transferase V(CyaA-GnT-V). The ability of recombinant CyaA to induce CTL responsesagainst these two epitopes in vivo was assessed in HHD mice, which aretransgenic for the human MHC class I molecule HLA*0201 and have beenshown to develop HLA*0201-restricted CTL responses against tumoralpeptides (26). HHD mice were immunized by 3 i.p. injections of 50 μg ofrecombinant CyaA with alum. After in vitro stimulation of splenocyteswith the corresponding peptide, CTL responses were tested in a chromiumrelease assay, using as targets peptide-pulsed RMA-S-HHD cells, whichexpress the same transgene as HHD mice. As shown in FIG. 1, bothrecombinant toxins carrying either Tyr or GnT-V epitopes induce strongCTL responses against target cells loaded with the relevant peptide.These CTL responses were antigen-specific since only peptide sensitizedtarget cells were killed and CTL activity was not detected on targetcells loaded with irrelevant peptides (data not shown). As expected, nosignificant CTL responses were observed in mice immunized with the wildtype CyaA showing that the induction of specific CTL responses requiredin vivo priming by the epitope inserted into recombinant CyaA.

Induction of CTL responses by CyaA-Tyr was then analyzed using differentimmunization protocols with or without alum as adjuvant. As illustratedin FIGS. 2A and B, two i.p. injections of CyaA-Tyr were enough to inducespecific CTL responses against the tyrosinase epitope, even in theabsence of alum. Induction of strong specific CTL responses was alsoobserved following a single injection of 50 μg of CyaA-Tyr withoutadjuvant using the i.v. route (FIG. 2C). As expected, these CTLresponses were observed only when using peptide pulsed target cells andsplenocytes from mice immunized with the recombinant CyaA-Tyr, showingthe specificity of the responses. These results demonstrate the highefficiency of CyaA-Tyr to induce specific CTL responses against thetyrosinase melanoma epitope. However, using similar conditions ofimmunization (2 or 1 i.p. injections with or without alum), no specificCTL response was observed with the recombinant toxin CyaA-GnT-V,indicating that this toxin is less efficient to generate specific CTLresponse than the CyaA-Tyr (data not shown). However, by the intravenousroute, one injection of CyaA-GnT-V was sufficient to induce a strong CTLresponse (FIG. 2E).

Finally, CTL responses induced by genetically detoxified mutants of CyaAcarrying Tyrosinase or GnTV epitopes that are devoid of adenylatecyclase activity following insertion of a dipeptide into the catalyticsite were analyzed. HHD mice immunized with these detoxified moleculesdeveloped specific CTL responses against both tyrosinase and GnTVepitopes (FIGS. 2D, 2F), which were comparable to the responses of miceimmunized with the toxic forms of CyaA carrying the correspondingepitope. These results indicate that HLA*0201-restricted CTL inductionis independent of the catalytic activity as it was clearly demonstratedfor a viral epitope from LCMV in BALB/C mice (18).

Example 3 Recombinant CyaA-Tyr Induces Long Lasting Memory CTL Responses

To analyze the persistence of the CTL responses induced by therecombinant CyaA bearing melanoma epitope, HHD mice received two i.p.injections of 50 μg of CyaA-Tyr in the presence of alum. Three and fivemonths after the last injection, splenocytes from immunized mice werestimulated in vitro over five days with the peptide pTyr and then, theircytolytic activity was tested against peptide pulsed RMA-S-HHD targetcells. As illustrated in FIG. 3, CyaA-Tyr induced a long-lastingspecific CTL response because specific cytotoxic activity could bedetected in all mice three months after the last injection, and evenafter five months in one animal.

Example 4 HLA*0201-Restricted Peptides Inserted into CyaA are Processedand Presented by HLA*0201⁺ Human DC

In vivo induction of specific CTL responses by recombinant CyaAindicates that inserted epitopes are efficiently processed and presentedby murine APC. However, it is important to demonstrate that human APCare also able to process and present these HLA*0201-restricted epitopesinserted into CyaA. Because DC are the most important APC to induceprimary T cell responses, the ability of HLA*0201⁺ DC incubated with therecombinant CyaA to stimulate human CTL clones specific for the epitopesinserted into the recombinant CyaA was determined. For theseexperiments, human DC were generated in vitro from HLA*0201⁺ adherentPBMC in the presence of GM-CSF and IL-4. Increasing doses ofCyaA-E5-Tyr, CyaA-E5-GnT-V or control CyaA-E5 were then added andpresentation of the antigenic peptides was assessed by measuring theability of the treated DC to stimulate the relevant CTL in an IFN-γproduction assay.

As shown in FIG. 4A, human DC incubated with CyaA-E5-Tyr induced a highproduction of IFN-γ by the tyrosinase-specific CTL clone, indicatingthat the tyrosinase epitope is efficiently processed and presented inassociation with HLA*0201 molecules. The specificity of this recognitionwas confirmed by the lack of stimulation of two irrelevant CTL clones(data not shown) and by the lack of stimulation of thetyrosinase-specific clone by DC treated with the control toxoid CyaA-E5(FIG. 4A). Presentation of the tyrosinase epitope was proportional tothe dose of CyaA-Tyr up to 30 nM. Under these conditions, higher dosesappeared to be toxic for the DC, as indicated by the low recognition ofthe treated DC and by their decreased ability to present the syntheticpeptide loaded exogenously (data not shown).

In order to assess the relative efficiency of antigen presentation usingCyaA as delivery system a titration curve of the tyrosinase syntheticpeptide, which was pulsed on similar DC, was also performed. As shown onFIG. 4A, the CyaA-Tyr was up to 100 times more efficient than thesynthetic peptide to induce the presentation of the epitope by DC.

Human DC incubated with CyaA-E5-GnT-V induced a weak but reproducibleproduction of IFN-γ by the GnT-V specific CTL clone, as compared with DCincubated with the peptide pGnT-V (FIG. 4B). This result indicates thathuman DC are able to present the GnT-V epitope inserted into CyaA,although with a moderate efficiency.

In summary, these results clearly demonstrate the capacity of human DCto process and present human epitopes inserted into CyaA.

Example 5 CyaA Toxin Constructions which do not Induce a Response

CyaA-Mel 21, which comprises the epitope gp100-280, and includes theinserted sequence YLEPGTVTA formed the GP 100 melanoma-associated tumorantigen, does not include a CTL response. Similarly, CyaA-CEA 13, whichcomprises the epitope CEA 571-579, and has the inserted sequenceYLSGANLNL from the Carcinoma Embryonic Antigen, does not induce a CTLresponse. Neither of these toxins induce a CTL response specific for theinserted epitopes in the HHD mouse. Furthermore, some human dendriticcells cannot present the inserted epitope CyaA-Mel 21 (epitopegp100-280) to a human CTL clone. Therefore, this toxin is probably alsoineffective in humans. These two toxins, CyaA-Mel21 and CyaA-CEA13, areidentical to CyaA-Tyr and CyaA-GnTV, but differ only in the insertedsequences. Therefore, only the epitopes are different, and so theresponse to CyaA-Tyr and CyaA-GNTV are epitope specific.

Finally, the Cya-Mage toxin, which comprises the Mage A10/A2 epitopewith the inserted sequence GLYDGMEHL from the melanoma protein Mage 10,induces very good CTL response in HHD mouse, but is inefficient inhumans under the protocol described above. Therefore, a positiveresponse in the mouse is not always indicative of a positive response inhumans. In general, human responses are epitope and species specific.

REFERENCES

The following references are cited herein. The entire disclosure of eachreference is relied upon and incorporated by reference herein.

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1. An immunogenic composition comprising a recombinant protein, whereinthe recombinant protein comprises a Bordetella CyaA, or a fragmentthereof, and a peptide that corresponds to a tumor antigen.
 2. Theimmunogenic composition as claimed in claim 1, wherein the BordetellaCyaA, or fragment thereof, and the peptide are genetically fused orchemically bound together.
 3. The immunogenic composition as claimed inclaim 1, wherein the Bordetella CyaA is detoxified.
 4. The immunogeniccomposition as claimed in claim 1, wherein the tumor is a melanoma. 5.The immunogenic composition as claimed in claim 1, wherein the tumorantigen is an HLA*0201 epitope.
 6. The immunogenic composition asclaimed in claim 5, wherein the HLA*0201 epitope is Tyr or GnT-V.
 7. Theimmunogenic composition as claimed in claim 5, wherein the HLA*0201epitope is region 369-377 of tyrosinase.
 8. The immunogenic compositionas claimed in claim 5, wherein the HLA*0201 epitope is from an intron ofthe N-acetylglucosominyl-transferase V gene.
 9. The immunogeniccomposition as claimed in claim 5, wherein the HLA*0201 epitope isYMDGTMSQV.
 10. The immunogenic composition as claimed in claim 5,wherein the HLA*0201 epitope is VLPDVFIRC. 11-22. (canceled)
 23. Amethod of treating a patient with cancer comprising (1) administering aimmunogenic composition to the patient, wherein the immunogeniccomposition comprises a recombinant protein, wherein the recombinantprotein comprises a Bordetella CyaA, or a fragment thereof, and apeptide that corresponds to a tumor antigen, and (2) inducing a T cellresponse in the patient.
 24. The method as claimed in claim 23, whereinthe T cell response is a CTL response.
 25. The method as claimed inclaim 23, wherein the cancer is a melanoma.
 26. The method as claimed inclaim 23, wherein the tumor antigen is an HLA*0201 epitope.
 27. Themethod as claimed in claim 23, wherein the HLA*0201 epitope is Tyr orGnT-V. 28-33. (canceled)
 34. A method of treating a patient with cancercomprising (1) administering an immunogenic composition to the patient,wherein the immunogenic composition comprises a vector expressing arecombinant protein, wherein the recombinant protein comprises aBordetella CyaA, or a fragment thereof, and a peptide that correspondsto a tumor antigen, and (2) inducing a T cell response in the patient.35. The method as claimed in claim 34, wherein the T cell response is aCTL response.
 36. The method as claimed in claim 34, wherein the canceris a melanoma.
 37. The method as claimed in claim 34, wherein the tumorantigen is an HLA*0201 epitope.
 38. The method as claimed in claim 34,wherein the HLA*0201 epitope is Tyr or GnT-V. 39-44. (canceled)